1. Field of the Invention
The invention relates to the field of single-fiber optical tweezers and scissors enabled in-depth multi-functional micro-manipulation of cells.
2. Description of the Prior Art
Optical scissors and tweezers have been tools of the biologist for over two decades. Laser scissors uses lasers to alter and/or to ablate intracellular organelles and cellular and tissue samples, and today have become an important tool for cell biologists to study the molecular mechanism of complex biological systems. Single cells or groups of cells have been perforated for injection of exogenous materials, induction of DNA damage in cells, micro-dissection of neuronal processes as well as other intra-cellular organelles such as chromosomes, microtubules. Clinically, it has also been used to reduce the thickness of the zona pellucida layer of the ovum in order to improve human in vitro fertility. In all these applications, either a scanning stage or scanning mirror was used to scan a region in a single cell or group of cells for micro-processing.
Optical manipulation of microscopic objects using spatially sculptured optical landscapes coupled with optical binding is gaining considerable interest for engineering self-assembled colloidal and biological structures. While far-field binding between microscopic objects has been demonstrated using elliptical beams or two counter propagating beams, near-field trapping and binding over a large area has been reported at the interface of total internal reflection (TIR) occurring in a prism. Except for two fiber trapping, all other approaches have depth limitation. The two-fiber configuration requires critical alignment of the two counter propagating beams and therefore restricts three-dimensional (3D) manipulation of the optically bound structure. Theoretical evaluation of the trapping force exerted by the microfocused beam from an axicon-tipped single fiber and its use for in-depth trapping of cells and low-index particles has been demonstrated recently. An axicon (having a conical distal or terminal surface) can be used to turn a Gaussian beam into a Bessel beam, with greatly reduced diffraction and smallest optical confinement zones. The micro-axicon fiber can trap at a larger distance from the fiber tip compared to a tapered fiber.
Recently, the application of optically based micromanipulation has led to an explosion of new applications. In particular, optical tweezers and scissors have had a major impact on the fields of biophysics and colloidal science with applications ranging from measurement of force at the single molecule level to disease diagnosis to therapeutic applications in the field of assisted reproductive therapy.
Recently, while optical tweezers have been shown to enhance and guide neuronal growth, femtosecond laser scissors have been employed for axotomy of neurons, allowing measurement of the regeneration process. In contrast to the short working distance of the high numerical aperture, NA, microscope objectives, optical tweezers and scissors based on a single optical fiber will enable micromanipulation at much larger depths and thus open up additional avenues for biophysics and nanoscience research.
While no report exists on single-fiber scissors, earlier attempts to trap in three dimensions using a single optical fiber have not been successful, even with a hemispherical lens built on the tip of fiber. This failure is presumably due to the dominance of the scattering force in the axial direction. While particle trapping using a single fiber probe with an annular light distribution required balance of opposing optical and electrostatic forces, recently, pure-optical 3-D trapping was demonstrated using a tapered and axicon-tip fiber.
The short working distance of microscope objectives have severely restricted the application of optical tweezers and scissors at larger depths. Therefore, there is a growing attention towards use of optical fibers for manipulation of microscopic objects. Recently, in-depth single fiber optic trapping of low as well as high index particles has been demonstrated using micro-axicon tip fibers. The shaping the axicon tip cone angle enabled fiber optic trapping in near-field. Further, we have demonstrated controlled guidance of neuronal growth cones as well as trapping and stretching of neurons using the fiber-optic tweezers. The cells could be stretched by combined action of two forces, an attractive gradient force due to fiber optic tweezers at high beam powers pulling the membrane and a scattering force on the membrane as reported in dual fiber trapping. Alignment of intra-cellular dark (high refractive index) material along the direction of laser beam propagation was also observed. By mode-locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes. This microscopic-controlled nano-dissection of neurons followed by a process of resealing and repair could serve as a useful tool for basic and applied studies on neuronal damage, repair and regeneration. At reduced average power of the femtosecond fiber-optic microbeam, microinjection of impermeable exogenous materials into the trapped cells was also possible. At high average powers, lysis of a three-dimensionally trapped cell was accomplished.